The Impact Of Foliar Salicylic Acid In Salt-Exposed Guava (Psidium Guajava L.) Seedlings

ABSTRACT The rising demand for guava fruit worldwide is due to its richness in vitamin C, high yielding and two bearing seasons. Given the salt stress threat to guavas and the necessity of protective strategies, in the present research guava seedlings were subjected to sodium chloride (0 and 50 mM) and salicylic acid (0 and 1 mM). To ascertain a logical conclusion, the activity of antioxidant enzymes, antioxidant capacity of DPPH, ferric reducing antioxidant power, total flavonoid and phenol contents were monitored in salt-exposed guava seedlings. Based on our findings, the most superoxide dismutase (87.46 µmol min−1g−1DW) activity, ferric reducing antioxidant power (408.82 mmol g−1 DW), DPPH scavenging activity (80.65%), flavonoid (14.15 mg QE−1 g DW) and phenol (34.02 mg GAE g−1 DW) contents were found in 50 mM NaCl-exposed plants, which were enriched by 1 mM salicylic acid. A high correlation was observed among non-enzymatic antioxidant parameters (DPPH scavenging activity, ferric reducing antioxidant power, total flavonoid content and total phenol content). In conclusion, salicylic acid improved guavas salt tolerance by increasing both enzymatic (peroxidase and superoxide dismutase) and non-enzymatic (ferric reducing antioxidant power, DPPH scavenging activity, total phenol and flavonoid contents) pathways.


Introduction
Salinity due to human-induced practices, changes in irrigation systems and rising temperature is growing in the most developing countries and becoming a serious concern which declines the productivity of sensitive crops (Munns and Tester, 2008). The ionic homeostasis, under high salt concentrations, leads to osmotic stress and consequently extra generation of reactive oxygen species (ROS) (Khan and Panda, 2007) that causes destructive oxidative stress, chlorophyll breakdown, lipid peroxidation, protein oxidation and finally DNA impairment (Terzi and Kadioglu, 2006). Salinity threatens plant production over at least 20% of irrigated lands, thus introducing of capable approaches to mitigate stress injury is of a great priority. Over recent decades, some efforts have been introduced as efficient strategies to lessen the destructive impacts of salt stress on plant metabolism, i.e. the exogenous applications of putrescine (Esfandiari Ghalati et al., 2020), nitric oxide (Kaya et al., 2019), proline (Freitas et al., 2019) and salicylic acid (Naser Alavi et al., 2014).
Foliar salicylic acid (pH of 3, the volume of 250 ml for every plant) was applied (for Plants related to the mentioned treatment) at the fourth and sixth week after the establishment of the seedlings. Salt (NaCl) application, through irrigation, was started from the 5 th week of plants establishment until the 7 th week (Cavalcante et al., 2007; Esfandiari Ghalati et al., 2020. NaCl application was started with 17.094 mM and slowly reached the final level (50 mM). Afterward, all experimental units were irrigated with distilled water for 8 weeks. Finally, the leaf blades of upper branches were collected for the following measurements.

The Antioxidant Enzymes Assay
About 0.5 g of the fresh leaf was powdered by liquid nitrogen and mixed with the extraction buffer (100 ml of 50 mM phosphate buffer with pH = 7, 1 g of Polyvinyl pyrrolidone (PVP) and 0.0372 g of EDTA). The mixture was centrifuged (16128 G force, 15 min, 4°C) (Dhindsa et al., 1981). The supernatant was used as the enzyme extract for following assays.
The peroxidase (POD) activity was assessed by Chance and Maehly (1995) protocol. Briefly, 33 µl of the enzyme extract was added to one ml of the peroxidase reaction solution (13 mM Guaiacol, 5 mM hydrogen peroxide and 50 mM phosphate buffer with pH = 7). Then the absorbance was recorded at 470 nm using a Cecil CE2501 spectrophotometer (Chance and Maehly, 1995).
The superoxide dismutase (SOD) activity was assayed via the method was described by Beauchamp and Fridovich (1971). Briefly, 50 μl of the enzyme extract was added to one ml of the SOD reaction solution (50 mM phosphate potassium buffer with pH = 7.8, 75 mM NBT, 13 mM L-methionine, 0.1 mM EDTA and 2 mM riboflavin) and incubated for 15 minutes (under fluorescence light). Then the absorbance was recorded at 560 nm (Beauchamp and Fridovich, 1971).

Assays of Total Flavonoid Content, Total Phenol Content, Ferric Reducing Antioxidant Power and DPPH Scavenging Activity
The leaf samples were powdered, then 0.5 g of the powder was soaked in 80% methanol and centrifuged (112000 G force, 10 min, 4°C). The supernatant was used as the leaf extract for the following assays.
The total flavonoid content (TFC) was measured by Beketov et al. (2005) procedure. The leaf extract (0.2 ml) and quercetin (25 µg ml −1 ) were mixed with 0.2 ml of 10% AlCl 3 in 0.1 ml of methanol, 33% acetic acid and 90% ethanol. The samples were incubated at room temperature for 30 minutes. The procedure was terminated by recording the absorbance at 414 nm. The total flavonoid content was expressed as mg g −1 of quercetin (QE) equivalent in milligram g −1 dry weight (mg QE −1 g DW) (Beketov et al., 2005).
The total phenol content (TPC) was assayed using the spectrophotometric technique (Spanos and Wrolstad, 1990). The reaction mixture was 10 µl of the methanol leaf extract, 500 µl of 10% Folin-Ciocalteu's reagent in water, 500 µl of 1% NaHCO 3 and 490 µl of distilled water. The samples were vortexed and incubated at 24°C for 12 hrs. Then, the absorbance was determined at 765 nm. Gallic acid (100-200 mg l −1 ) was used to prepare the standard curve. The total phenolic content was expressed in mg Gallic acid equivalent g −1 DW (mg of GAE g −1 DW).
The Benzie and Strain (1999) technique was used for the ferric reducing antioxidant power (FRAP) assay. The first step was the preparation of 300 mM acetate buffer (3.1 g of CH 3 COONa and 16 ml of CH 3 OOH, pH = 3.6), 20 mM FeCl 3 · 6H 2 O and 10 mM TPTZ (2, 4, 6-tripyridyl-s-triazine) solutions in 40 mM HCl. The second step was the preparation of FRAP solution by mixing 25 ml of the acetate buffer, 2.5 ml of FeCl 3 · 6H 2 O and 2.5 ml of TPTZ. The next step involved mixing 150 μL of the leaf extract with 2850 μl of the FRAP solution and allow to react under dark conditions at 37°C for 30 min. The final step was recording the optical density of the extracts at 593 nm. The standard curve was prepared using 50-1000 μM FeSO 4 . The results were expressed in mmol g −1 dry weight (Benzie and Strain, 1999).
For the DPPH scavenging activity assay, the leaf extract (50 μl) was mixed with 950 μl of 0.1 N DPPH (1,1-diphenyl-2-picryl-hydroxyl). The samples were kept under dark conditions (24°C, 30 min). Then, the absorbance was recorded at 517 nm. The free radical scavenging activity was calculated using the following equation (Singelton et al., 1999): DPPH scavenging activity (%) = ((A cont -A samp ) ×100)/A cont Where A cont and A samp are the absorbance values of the standard and the sample.

Data Analysis
The SAS version 9.1.3 ("SAS® Procedures, 1990) was used for data analysis. The research was a factorial experiment in a completely randomized block design with 6 replications. The factors were salicylic acid and salinity levels. The data normality was tested with Shapiro-Wilks test. The homogeneity of variance was confirmed with the Pillai's trace test. The mean comparisons were computed by Tukey's test (P < .01). The correlation analysis was used to determine parameters relations (Pearson coefficient). The graphs were figured using Sigma Plot (n.d, 10.0).

Results
The analysis of variance showed that the studied parameters had a significant behavior, due to NaCl treatment, salicylic acid application and their interaction ( Table 1).

Interaction of Salt and Salicylic Acid on POD and SOD Activities
According to our findings, the POD activity was reduced by salt treatment, compared to control (saltfree and no salicylic acid usage). Maximum POD activity (0.42 µmol min −1 g −1 DW) occurred in the control plants which were received SA treatment. Besides, the application of 1 mM salicylic acid significantly caused an increase in POD activity. The lowest POD activity (0.25 µmol min −1 g −1 DW) was followed by 50 mM NaCl under SA-free conditions ( Figure 1A). Treating with 50 mM NaCl increased the SOD activity (12.06% increment compared to control) and reached 70.58 µmol min −1 g −1 DW. Salicylic acid, in salt-free plants, caused a rise in SOD activity (4.88% increase). The highest level of SOD activity (87.46 µmol min −1 g −1 DW) was found in 50 mM NaCl with 1 mM salicylic acid ( Figure 1B).

Interaction of Salt and Salicylic Acid on FRAP and DPPH Capacity
NaCl significantly increased the ferric reducing antioxidant power (FRAP) in guava leaves (5.3% increase); supplementary salicylic acid also caused an increase in FRAP (0.25% increase). As illustrated in Figure 3A, the most FRAP value was obtained in the plants which had received both NaCl and salicylic acid (408.82 mmol g −1 DW). The minimum FRAP value (327.89 mmol g −1 DW) was observed in the control plants (Figure 2A). Both NaCl and salicylic acid made an increase in DPPH capacity (59.50% and 10.76% upturn, respectively). The most DPPH value (80.65%) was found with 50 mM NaCl enriched by salicylic acid (1 mM). The minimum value (26.67%) was observed in the control plants ( Figure 2B).

Interaction of Salt and Salicylic Acid on TFC and TPC
The application of both NaCl and salicylic acid resulted in an increase in TFC. The minimum flavonoid content (10.56 mg QE g −1 DW) was detected in the control plants, which was not significantly different from those received SA (11.09 mg QEg −1 DW). The most value (14.15 mg QE g −1 DW) was related to 50 mM NaCl and SA treated plants ( Figure 3A).
According to our data, salt made an 14.85% increase in TPC in comparison with the control plants (no salt, SA-free). However, the application of 1 mM salicylic acid non-significantly increased TPC in the control plants (3.95% raise). The most TPC value was related to the combination of both 50 mM NaCl and 1 mM salicylic acid (34.02 mg GAE g −1 DW). The minimum value occurred in the control plants (24.28 mg GAE g −1 DW) ( Figure 3B).

Discussion
Abiotic stresses cause the interruption of electron transport chains in mitochondria and chloroplasts of plants. Under such conditions, O 2 donates an electron and rises the ROS accumulation (Grob et al., 2013). The plant defense response is an extra-activation of some antioxidant enzymes and deactivation of some others to protect the macro-molecules (Ahmed et al., 2009). SOD is the primary enzyme in the detoxification mechanism, which converts superoxide FRs to H 2 O 2 (Sairam et al., 2002). The overproduction of H 2 O 2 reduces the membrane permeability and leads to lipid peroxidation and oxidative stress. Thus, the generated H 2 O 2 is scavenged by catalase, ascorbate peroxidase and glutathione peroxidase (Hamanaka and Chandel, 2009). Our results about the increased activity of SOD under salt stress, are in line with those in mulberry (Sudhakar et al., 2001) and pistachio (Lotfi et al., 2015). Also, we identified a decrease in the POD activity in salt-exposed plants. This decline is related to the distracted scavenging activity of this enzyme (Shahbazi et al., 2011). This finding was in line with those of Ahmad Sajid et al. (2016), Yordanova and Popova (2007) and Esfandiari Ghalati et al. (2020). SA adjusts the antioxidant capacity of the salt exposed-plants via increasing or inhibiting the activity of antioxidant enzymes including catalase, superoxide dismutase and ascorbate peroxidase (Ahmad et al., 2018;El-Esawi et al., 2017;Li et al., 2014;Pirasteh-Anosheh and Emam, 2018). Also, exogenous application of salicylic acid encourages the expression of the genes encoding the  antioxidant enzymes (Farooq et al., 2009;Horvath et al., 2007). Exogenous SA has improved the activity of POD (War et al., 2011) and PPO (Schneider and Ullrich, 1994) enzymes. Foliar applied SA boosted the activity of superoxide dismutase and peroxidase in tomato  and mustard plants (Yusuf et al., 2008). Salinity prompts the generation and accumulation of reactive oxygen species (Khan and Panda, 2007). The changes in antioxidant levels modulate ROS scavenging mechanisms (Mittler, 2002). Hence, plants have equipped with several protection mechanisms, including the biosynthesis of phenolic and flavonoids compounds, against harmful ROS (Koyro et al., 2012). Phenolic compounds, the plant's secondary metabolites, are synthesized via both shikimic acid and phenylpropanoid metabolic pathways. In plants, these compounds might act as signaling molecules, motivate disease resistance and protect against reactive oxygen species, produced by abiotic stresses such as salinity (Crozier et al., 2006). The role of phenolic compounds as an anti-oxidative agent and their increment due to salt stress has already reported (Chaparzadeh and Hosseinzad-Behboud, 2015;El-Esawi et al., 2017).
The accumulation of ROS under unfavorable environmental conditions, shifts to increase the plant hormones, such as salicylic acid (Ahlfors et al., 2004). Induced salicylic acid, as well as the exogenously applied form, provokes the synthesis of secondary metabolites (Idrees et al., 2010;Ghasemzadeh et al., 2012;Pirasteh-Anosheh and Emam, 2018). Actually, when applied in low concentrations, salicylic acid might cause a temporary and non-serious oxidative stress, which turns the hardening process in plants, improves the anti-oxidative ability of the plants (Knörzer et al., 1999) and hence assists in the biosynthesis induction of phenolic compounds (Németh et al., 2002). There are documents of increased plant antioxidant capacity in saline conditions after SA treatment (Chaparzadeh and Hosseinzad-Behboud, 2015;Farhangi-Abriz and Ghassemi-Golezani, 2018;Gorni et al., 2019;Grzeszczuk et al., 2018;Li et al., 2014). Also, exogenous SA is found to boost the production of plant flavonoids through increasing the activity of core flavonoid biosynthetic genes (Xu et al., 2009), which are in line with our results.
Correlation analysis examines the relationships between variables (Yadav, 2018). As previously reported by Rodriguez-Dominguez et al. (2016), salt stress modifies cell wall integrity and leaf turgor, lead to a decrease in photosynthesis rate and leaf number. Such changes alter physiological processes and the relationships between the growth parameters under different conditions. According to correlation analysis, a high correlation was observed among non-enzymatic antioxidant parameters (DPPH, FRAP, TPC, TFC).

Conclusion
Based on our findings, NaCl made a reduction in POD activity, while an increment in SOD activity, TPC, TFC, FRAP and DPPH capacities in guava plants. Salicylic acid improved the antioxidant ability of salt-exposed guavas by increasing the SOD, POD, FRAP, DPPH, TPC and TFC. The effects of salicylic acid in mitigating oxidative stress suggest the SA application as a potential, simple and efficient approach to diminish the injury of oxidative stress in salt-exposed guavas.